1. Field of the Invention
The present invention relates to a method of preparation of transgenic plants resistant to viral infections, a recombinant vector and so obtained transgenic plants.
2. Description of the Related Art
Many plant diseases are caused by RNA viruses, such as, e.g., tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), potato virus (PV), and so forth. Most viral infections reduce crop yields and, sometimes, may even cause catastrophic effects causing considerable financial losses.
CMV, a virus belonging to Cucumovirus type, is diffused in nature by flying carriers, i.e., aphids. From these, at least 60 different species are capable of collecting the virus from an infected plant, and retransmitting it directly to a healthy plant, without a lag period inside the insect (non-persistent transmission).
CMV is a polyphagous virus, certainly from phytoviruses, the one with the widest ascertained range of hosts, approximately a thousand of different species (Palukaitis et al., Adv. In Virus Res. 41:281-348, 1992). It derives that the inoculum is always present in nature, both in wild and cultivated plants, acting as reservoirs from which aphids collect their virus supply.
The disease caused by CMV manifests with different symptoms: reduced development of the plant, foliar chlorosis and several malformations both in aerial parts of the plant and in fruits. However, the most serious pathology, caused by some necrogenic viral strains, appears in tomato and is given the name of "lethal necrosis".
From a molecular viewpoint, CMV displays a tripartite RNA genome, arranged inside the coat protein (CP). Genomic RNA's, which are single-stranded and have mRNA(+) polarity, are classified, with reference to their molecular weight, into RNA-1 (3.4 Kb), RNA-2 (3.0 Kb) and RNA-3 (2.4 Kb). Furthermore, in viral preparations, a fourth RNA is present which encodes the coat protein, is referred to as "sub-genomic RNA-4" of 1,027 Kb from RNA-3 and is co-linear with this.
RNA 1 and 2 are monocistronic and encode two proteins, respectively of 991 and 839 aminoacids, correlated with virus replication activity. RNA-3 is dicistronic and, at its 5' terminal region encodes a protein implied in virus transportation into the plant, and at its 3'-terminal region contains the genes encoding the coat protein (CP). In intercistronic region of RNA-3 the sub-genomic promoter is present which is important in vivo for the formation of sub-genomic RNA-4 (+) on which the coat protein is translated. In fact, replicase recognizes the promoter sequence on (-) strand of RNA-3, thus securing the synthesis of RNA-4.
In some viral extracts, a small extragenomic RNA is furthermore present, which is known as "RNA-5" or, more commonly, CARNA-5, belonging to the class of satellite RNAs. CARNA-5 performs an important biologic function because it is capable of modulating the symptoms of CMV infection, and namely, rendering them either lighter or more serious. In fact, two types of CARNA-5 exist, which are referred to as "necrogenic" the one, and "non necrogenic" the other (also said "benign"), owing to its capability of inducing, or less, necrotic symptoms in infected plants.
Some valuable agricultural practices, as rotation of crops and removal of weeds and crop's residues or use of insecticides, may be able to control the viral attack, but only partially. In fact, a fast assay bite on a plant treated with insecticides is enough to infect it, also if the aphid subsequently dies.
Therefore, alternative solutions were proposed in the art for combatting phytoviruses, in general, and CMV in particular, which are essentially based on the introduction of non-conventional resistances, or transgenic resistance, and comprise:
1) Using benign variants of CARNA-5 for a plant pre-immunization or vaccination. PA0 2) Introduction of satellite RNA into the genome of the plant PA0 3) Use of the gene encoding the coat protein. PA0 4) Use of antisense RNA
This method consists in infecting the plants simultaneously with the pathogenic virus and benign satellite RNA in order to create a tolerance to this virus. The variability of the compounded viral genomes and the high recombination frequency, which may be limited only by means of a control of the vaccination system which is very laborious and burdensome, display however potential risks. In fact, the virus used for the vaccination may possibly diffuse into the surrounding environment, in which it could cause synergisms with other pathogens, thus causing damages to more sensitive species. Therefore, this method cannot be applied on large areas.
In order to avoid the problems derived from preinoculation, the plants have been modified by adding satellite RNA to their genome. In the resulting plants, a reduction of symptoms caused by virus could be observed.
However, the genetic transformation with satellite RNA does not exclude the risk of a back mutation. A necrogenic variant of CARNA-5 possibly arising could be rapidly diffused throughout the surrounding environment through the natural infection ways, with serious consequences. Furthermore, owing to the satellite-virus-plant interaction, apparently "benign" satellites could cause damages to different plants from source plants.
Recently, it was demonstrated for different viral species with tripartite or monopartite RNA, that, through the introduction into plants of RNA-4 gene encoding the coat protein, transgenic plants can be obtained which display a strong decrease in disease symptoms when they are exposed to infections with the same virus.
However, also this method is not free from drawbacks. In fact, the coat protein produced inside the vegetable tissues can encapsidate foreign RNA to virus (heterologous encapsidation), thus originating new viral particles with can be acquired and diffused within the surrounding environment by the carriers (Palukaitis et al., Adv. In Virus Res. 41:281-348; 1992).
Another strategy used in order to obtain transgenic plants resistant to determined viruses consists in inserting into the plant a DNA sequence which is complementary to a portion of the viral genome in antisense orientation (not encoding). Unfortunately, the use of such a strategy gave unsatisfactory results. In fact, transgenic tobacco plants, obtained by using the antisense gene of the coat protein of CMV and PVX, displayed a tolerance to virus only when they were infected with low inoculum concentrations (Cuozzo et al., Biotechnol., 6:549-557, (1988); Hemenway et al., EMBO J. 7:1273-1280, 1988). Furthermore, discouraging results were obtained when antisense genes were used which were capable of complementing with different domains of genomic RNAs of CMV. In fact, only in one case low resistance levels were observed (Rezaian et al., Plant Molecular Biology, 11:463-471, 1988).